Method and reagent for sulfurization of organophosphorous compounds

ABSTRACT

A composition suitable for sulfurizing an organophosphite. The reagent comprises a solution of sulfur and a tertiary amine, in a suitable solvent such as carbon disulfide. The tertiary amine is preferably an optionally substituted trialkyl amine, for example triethylamine or diisopropylamine. A method is also provided in which phosphites or phosphonous esters are sulfurized to their corresponding phosphorothioates or phosphonothioates, respectively, using compositions of the present invention. This method has particular application to produce internucleotide phosphorothioate or phosphonothioate bonds in a nucleotide multimer.

FIELD OF THE INVENTION

This invention relates to the sulfurization of phosphites andphosphonous esters. In another aspect it relates to phosphorothioateanalogs of nucleic acids. In another aspect it relates to methods forsynthesizing such analogs. In a further aspect it relates to reagentsuseful in the sulfurization of phosphites and phosphonous esters, forexample, the synthesis of phosphorothioate analogs of nucleic acids.

BACKGROUND OF THE INVENTION

Nucleic Acids occur in nature as chains of either ribonucleotides ordeoxyribonucleotides, the individual nucleotides being linked to eachother by phosphodiester bonds between the ribose and deoxyribose sugarswhich form, respectively, the backbones of ribonucleic acids (RNA) ordeoxyribonucleic acids (DNA). Apart from their role in naturallyoccurring phenomena, DNA and RNA, particularly DNA and RNAoligonucleotides, are expected to play an increasingly important role inmedical diagnostic and therapeutic applications. For example,oligonucleotides have been shown to be useful in a variety of "probe"assays for viral and bacterial diseases and for the detection of geneticabnormalities. In these assays, the "probe" is typically anoligonucleotide selected to be complementary to a RNA or DNA sequencewhich is unique to the organism or genetic defect to be detected(Matthews et al., Anal. Biochem., Vol. 169, (1988)).

It has also been observed that oligonucleotides which are complementaryto messenger RNA (antisense oligonucleotides) can be introduced to acell and arrest the translation of the mRNA. This arrest is believed toresult from the hybridization of the antisense oligonucleotide to themRNA. See, for example, Stephenson, et al., Proc. Natl. Acad. Sci., USA,75, 285 (1978) and Zamecnick, et al., Proc. Natl. Acad. Sci., USA, 75,280 (978).

The ability of antisense oligonucleotides to inhibit or prevent mRNAtranslation suggests their application in antiviral therapy. A virusinfecting a cell reproduces its genetic information by using thebiological machinery of the infected cell. Transcription and translationof that information by the cellular ribosomes are essential to viralreproduction. Thus, if expression of the viral gene can be interrupted,the virus cannot replicate or may replicate at such a slow rate as togive the host's immune system a better opportunity to combat theinfection.

It has been proposed to use oligonucleotides in viral therapy bydesigning an oligonucleotide with a nucleotide sequence complementary toa sequence of virally expressed mRNA which must be translated if viralreplication is to be successful. Introduction of the antisenseoligonucleotide to the cell permits it to hybridize with and prevent, orat least inhibit, this essential translation.

Conventional phosphodiester antisense oligonucleotides have beenreported to exhibit significant shortcomings as antisenseoligonucleotides. One limitation is that they are highly subject todegradation by nucleases, enzymes which breakdown nucleic acids topermit recycling of the nucleotides. In addition, most cells arenegatively charged. As a result, a phosphodiester oligonucleotide doesnot readily penetrate the cell membrane because of the density ofnegative charge in the backbone of the oligonucleotide.

It has been proposed to modify oligonucleotides to overcome theseshortcomings. One such proposal has been to use non-polar analogs ofconventional phosphodiester oligonucleotides. Such analogs retain theability to hybridize with a complementary sequence and would be expectedto enter the cell more readily and be less resistive to nucleasedegradation. Promising results have been obtained with methylphosphonate analogs. See Agris et al., Biochemistry 25, 1228 (1986).More recently phosphorothioate analogs, i.e., nucleic acids in which oneof the non-bridging oxygen atoms in each inter-nucleotide linkage hasbeen replaced by a sulfur atom, have also been shown to possess theability to block mRNA translation. In at least one case, inhibition ofexpression of the chloramphenicolacetyltransferase gene, aphosphorothioate analog has been shown to be superior to the methylphosphonate analog which in turn was shown to be more effective than theunmodified phosphodiester oligonucleotide. Inhibition of HIV virusreplication by a thiophosphorate analog has also been demonstrated. SeeMatsukara et al, Proc. Natl. Acad. Sci., USA, 84, 1 (1987).

Phosphorothioate analogs of oligonucleotide probes are also useful asreplacements for conventional probes for diagnostic and therapeuticapplications. However, only a few techniques have been reported for thesynthesis of phosphorothioate analogs of nucleic acids, all of themcumbersome and not well adapted for use with currently availableautomated nucleic acid synthesizers.

One reported synthetic technique, for example, uses presynthesizednucleotide dimers. The synthesis of the full array of sixteen dimersnecessary for the procedure is both laborious and expensive. (Connoly etal., Biochem., 23, 3443 (1984).

A more preferred procedure would permit use of the highly reactive,commercially available nucleoside-phosphoramidite monomers currentlyemployed with nucleic acid synthesizers. Such monomers are actually usedin processes for preparing phosphorothioate analogs. However, thesulfurization of phosphorous in the phosphite intermediate has been verytroublesome. For example, elemental sulfur in pyridine at roomtemperature requires up to 16 hours to produce the internucleotidephosphorothioate triester 12 as shown in FIG. I. (P. S. Nelson, et al.,J. Org. Chem., 49, 2316 (1984); P. M. S. Burgers, et al., Tet. Lett.,40, 3835 (1978)). A similar procedure using elemental sulfur, pyridineand carbon disulfide permitted sulfurization to be done at roomtemperature within 2 hours. B. A. Connolly, et al., Biochem., 23, 3443(1984). The triester is convertible to the phosphorothioate by basecatalyzed removal of substituent "R."

Carrying out the sulfurization at 60° C. in 2,6-lutidine requires 15minutes during automated, solid phase synthesis of phosphothioates fromCompound 11. W. J. Stec et al., J. Am. Chem. Soc., 106, 6077 (1984).However, most automated synthesizers do not have provisions for heatingthe column required for performing sulfurization at elevated temperatureand vaporization of the solvent at 60° C. would be expected to formbubbles in delivery lines which would reduce flow rates and even causesynthesis failures. Furthermore, even a fifteen minute reaction time forsulfurization after the addition of each nucleotide makes the procedurefar from optimal.

Accordingly, there has gone unmet a need for a process for thepreparation of phosphothioate oligonucleotide analogs that is rapid andthat lends itself to use on conventional DNA synthesizers.

TERMINOLOGY

The following terms are used in this disclosure and claims:

Nucleotide: A subunit of a nucleotide acid consisting of a phosphategroup, a 5 carbon sugar ring and nitrogen containing purine orpyrimidine ring. In RNA the 5 carbon sugar is ribose. In DNA, it is2-deoxyribose. The term also includes analogs of such subunits.

Nucleotide multimer: A chain of two or more nucleotides linked byphosphorodiester or phosphonodiester bonds, or analogs thereof.

Oligonucleotide: A nucleotide multimer generally about. 10 to 125nucleotides in length, but which may be greater than 125 nucleotides inlength. They are usually obtained by synthesis from nucleotide monomers,but may also be obtained by enzymatic means.

Nucleotide multimer probe: A nucleotide multimer having a nucleotidesequence complementary with a target nucleotide sequence containedwithin a second nucleotide multimer, usually a polynucleotide. Usuallythe probe is selected to be perfectly complementary to the correspondingbase in the target sequence. However, in some cases it may be adequateor even desirable that one or more nucleotides in the probe not becomplementary to the corresponding base in the target sequence, or thatvarious moieties of synthetic origin either replace a nucleotide withinthe probe or be inserted between bases of the probe. Typically, theprobe is labeled when used for diagnostic purposes.

Oligonucleotide probe: A probe of synthetic or enzymatic origin usuallyhaving less than about 125 nucleotides, but which may contain in excessof 200 nucleotides.

Hybridization: The formation of a "hybrid", which is the complex formedbetween two nucleotide multimers by Watson-Crick base pairings betweenthe complementary bases.

SUMMARY OF THE INVENTION

The present invention then provides a reagent which is suitable for theconvenient and efficient sulfurization of organic phosphites (i.e.trivalent phosphorus bonded to three oxy groups with at least onethereof being an organic-oxy group) and organic phosphonous esters (i.e.trivalent phosphorous bonded to only two oxy groups), to form thecorresponding thiophosphorus acid derivatives (specifically,phosphorothioates or phosphononothioates, respectively). The inventionis particularly suited to sulfurizing a suitably protectedinter-nucleotide, either 3'-5' or 2'-5' phosphite or phosphonouslinkages contained in oligonucleotides and ribonucleotide multimers insolution or on a solid support for the manual or automated synthesis ofphosphothioate oligonucleotides.

Accordingly, the present invention provides, in one aspect, a reagentand process using that reagent suitable for adding "sulfur" to aphosphite or phosphonous intermediate such as the kind 11 (see FIG. 1)to produce a phosorothioate or phosphonothioate of the kind 12 (see FIG.1). It should be noted that for compounds 11 the inter-nucleotidephosphite or phosphonous bond (and the inter-nucleotide phosphorothioateor phosphonothioate bond of 12) is shown between the 3'-hydroxyl of onenucleoside and the 5'-hydroxyl group of another nucleoside. However,those bonds can be between any two hydroxyl groups of two nucleosides,for example, between the 2' and 5' hydroxyls, or between the hydroxylgroup of a nucleoside and that of a non-nucleoside moiety or evenbetween two non-nucleoside moieties. The reagent comprises a mixture ofelemental sulfur, a solvent for sulfur, and a tertiary alkyl amine. Thepreferred reagent is:

0.2M elemental sulfur in a mixture comprising 50% carbon disulfide and50% diisopropylethylamine.

Other reagents of the present invention include:

(i) 0.2M elemental sulfur in a mixture comprising 50% carbon disulfideand 50% triethylamine;

(ii) 0.2M elemental sulfur in a mixture comprising carbon disulfide(25%), pyridine (25%) and triethylamine (50%).

The above reagents can rapidly sulfurize the phosphite or phosphonousintermediates of the kind 15 at room temperature in just about 45seconds, eliminating the need to choose between extended reaction timesrequired for the sulfurization step if it is to be done at roomtemperature, or the use of elevated temperatures to achieve more rapidsulfurization as required by prior art processes. FIG. 3 illustrates asulfurization process according to the invention as embodied in thesynthesis of phosphorothioate analogs of oligonucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sulfurization of an oligonucleotide phosphite orphosphonous ester to form a phosphorothioate triester, orphosphonothioate diester, of a nucleic acid, some preferred substituentsinclude: B is a purine or pyrimidine base; R₃ is either CH₃, OCH₃, orOCH₂ CH₂ CN; R₄ is either H, O-Silyl group, COCH₃ or COC₆ H₅ ; R₅ iseither a mono- or dimethoxy triityl group; and R₆ is either a long chainalkylamino silica support, COCH₃, COC₆ H₅, silyl group or atetrahydropyranyl group.

FIG. 2 illustrates the formation of an oligonucleotide phosphitetriester or phosphonous diester useful in the invention, with somepreferred substituents include: R is either CH₃, CH₂ CH₃ or i-Pr; and B,R₃, R₄, R₅ and R₆ are as described above.

FIG. 3 illustrates assembly of an oligonucleotide phosphorothioate,involving sulfurization of oligonucleotide phosphites using a reagent ofthe present invention and its conversion to the correspondingphosphorothioate.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As described above, the reagent of the present invention comprises amixture of elemental sulfur, an organic solvent for sulfur, and anon-aromatic tertiary amine. By a "non-aromatic" amine is meant an aminewhich is not a member of an aromatic ring. However this term does notexclude an aromatic group bonded to the nitrogen of an amine group. Thesolvent for sulfur can be selected from solvents such as carbondisulfide, toluene, benzene or ethyl alcohol. Carbon disulfide ispreferred as it is a particularly effective solvent for sulfur. Themixture can optionally include other substances, for example aromaticamines, such as pyridine. The useful tertiary amines include, forexample, triethylamine and diisopropylethylamine. Diisopropylethylamineis preferred.

The composition of the reagent can vary over a wide range and theparticular formulation which yields the best results can depend upon thephosphite or phosphonous ester being sulfurized. However, the mosteffective formulation of the reagent for a specific reaction can bedetermined by varying the components of the mixture. In addition,particular formulations may be found practically difficult to work with.For example, it was found that in a formulation in which1,8-diazabicyclo[5,4,0]undec-7-ene was used as the tertiary amine,sulfur precipitated out from the carbon disulfide solution.

The reagents of the present invention, then, are useful for thesulfurization of organic phosphites or phosphonous esters to form thecorresponding phosphorothioate. The starting organophosphorus compound,and its corresponding phosphorothioate produced by the method of thepresent invention, have the formulae: ##STR1##

In the above formulae, R₁ R₂, and R₃ may be the same or different andare preferrably selected from organic moieties such as optionallysubstituted alkyl, alkoxy, phenyl, phenoxy, and tertiary amino, andanalogues of the foregoing. In particular, R₁ and R₂ may beribonucleosides and deoxyribonucleosides.

The reagents of the present invention are particularly useful in thesynthesis of phosphorothioate analogs of oligonucleotides from aphosphite or phosphonous ester in which R₁ and R₂ are nucleosides,particularly suitably protected nucleosides. In the case where it isdesired to synthesize simply a phosphorothioate analog of a nucleotidemultimer, then R₃ is a group which can be selectively removed (cleaved)following sulfurization. Examples of such groups include methoxy, andβ-cyanoethoxy. However, if it is desired to produce a phosphonothioateanalog of a nucleotide multimer (i.e., an analog in which a phosphonouslinking group has an O═ replaced with S═), then R₃ need not be a groupwhich can be selectively removed following sulfurization. For example,R₃ could be methyl. Phosphites and phosphonous esters of the foregoingtype are readily obtained using the processes of U.S. Pat. No. 4,625,677and U.S. Pat. No. 4,458,066 (the disclosures of these patents, and allother references cited herein, being incorporated into this applicationby reference).

FIG. 2 illustrates the assembly of a dinucleoside phosphite orphosphonous ester 15 from compounds of the type 13 (phosphoramidites orphosphonamidites) and 14 (nucleotides). However, it will be appreciatedthat higher oligomers can be obtained by sequential additions ofphosphoramidites according to known procedures. Sulfurization ispreferably carried out after the addition of each nucleoside althoughsulfurization may be deferred and carried out on the assembled oligomer.A preferred reagent for the sulfurization of phosphites or phosphonousesters, particularly the internucleotides obtained from thephosphoramidite process, is 0.2M elemental sulfur dissolved in an equalvolume mixture of carbon disulfide and diisopropylethylamine.

Examples 1 and 2 below describe in detail the use of a sulfurizingreagent of the present invention for automated synthesis ofphosphorothioates of a different nucleotide multimers. Examples 3 and 4describe the use of a different sulfurizing reagent of the presentinvention for automated synthesis of nucleotide multimers. Examples 5and 6 are similar to Examples 1 through 4 but illustrate the use ofdifferent reagents. Example 7 describes the use of a further sulfurizingagent of the present invention for automated synthesis of variousphosphorothioate nucleotide multimers, while Example 8 is similar toExample 7 but describes the use of different reagents. Examples 9through 11 also describe the use of sulfurizing reagents of the presentinvention in the automated synthesis of various nucleotide multimers.Example 12 illustrates the Tm (melting temperature) of hybrids formed bythe hybridization of phosphorothioate analogs with a complementary RNAsequence.

EXAMPLE 1

Automated Synthesis of Phosphorothioate Analog of TTTT

FIG. 3 illustrates the scheme for the synthesis of the phosphorothioateanalog of TTTT

Materials: Gold label elemental sulfur, carbon disulfide anddiisopropylethylamine ("DIEA") were purchased from Aldrich FineChemicals, Milwaukee, Wis. Nucleoside phosphoramidites, 15-umolT-column, Tetrazole and Cap B (10% dimethylamino pyridine (DMAP) intetrahydrofuran (THF) were purchased from Biosearch, Inc., San Rafael,Calif. Cap A (10% acetic anhydride (AC₂ O) in THF) was prepared fromdistilled acetic anhydride (Aldrich). 2.5% trichloroacetic acid indichloromethane was prepared from gold label trichloroacetic acid(Aldrich). Acetonitrile, dichloromethane and tetrahydrofuran, allcontaining less than 0.002% water were purchased from J. T. Baker.

The scheme of FIG. 3 outlines the steps involved in the automatedsynthesis of a nucleotide tetramer involving automated sulfurization ofthe phosphite intermediate 19. Synthesis was performed on Bio-search8750 DNA synthesizer using a modified version of the standard synthesiscycle. This modification makes use of an aqueous wash (10% water in 2%pyridine-THF) step prior to the oxidation step. This modification wasincluded to minimize side reactions in the overall synthesis.Sulfurization was achieved by delivering a 0.2 molar solution ofelemental sulfur in 50% carbon disulfide/50% diisopropylethylamine, (allpercentages by volume unless otherwise indicated) for 15 seconds andletting it stand in the 15 μmol T-column for 30 seconds. After thesulfurization, the column and the delivery lines were washed withacetonitrile before beginning the next cycle of addition of the nextbase of the sequence.

As shown in FIG. 3, the synthetic procedure is initiated using5'-O-dimethoxytrityl-thymidine linked to controlled pore glass (CPG),reagent 16, as described, for example, in U.S. Pat. No. 4,458,066. The5'-dimethoxytrityl (DMT) protecting group is removed using acidcatalysis to form the deprotected intermediate 17 which is coupled tothe thymidine phosphoramidite 18 to form dimer 19. Any unreacted 17 iscapped by treatment with acetic anhydride and dimethylaminopyridine. Thedimer 19 is sulfurized with the same sulfurization reagent consisting of0.2M sulfur in 50% CS₂ /50% DIEA to form the phosphorothioate triester20. The 5'-protecting group is then removed as before to form dimer 21and the trimer (not shown) assembled using the thymidine phosphoramidite18 which is then sulfurized and deprotected, followed by assembly of atetramer using thymidine phosphoramidite 18. Sulfurization of thetetramer and removal of the 5'-protecting group in the same manner asdescribed, provides tetramer 22.

After tetramer 22 had been assembled, the resin from the column wastransferred into a screw-cap tube and heated with concentrated ammoniumhydroxide at 55° for 8-10 hours to form 23. Ammonium hydroxide was thenevaporated off to give 300 OD units of the product. An analyticalexamination of the purity was performed by gel electrophoresis of 2 ODunits of the crude product on a 10% acrylamide gel containing 7M urea.The product was largely one band (ca 95%) as shown by UV shadowing.

The extent to which the "sulfur" had been added to the "phosphorus" ofthe phosphite intermediate was determined by ³¹ P nuclear magneticresonance (NMR) in D₂ O. Absence of ³¹ P resonance at -2.688 PPM in theNMR spectrum of the phosphorothioate analog of T-T-T-T (³¹ P, 53.866PPM) indicates that the extent of sulfurization is almost quantitative.

EXAMPLE 2

The use of the sulfurization reagent was then extended to the synthesisof a phosphorothioate analog of a longer nucleotide multimer. Thislonger multimer was synthesized and deblocked the same way as describedin Example 1 but purified by gel electrophoresis (20% acrylamide--7Murea). The product was visualized though UV shadowing, the slow movingband was sliced off and extracted with 0.1M ammonium acetate (pH 7.2)and desalted on sep-pak followed by a second desalting on Pharmacia'sNap-25 column. Such a desalted multimer was labeled with δ-ATP³² and gelelectrophoresed to check the analytical purity. The followingphosphorothioate multimer was synthesized (the designation "PS" beforethe 5' end of the multimer sequence indicating the phophorous of allnucleoside linking groups has been sulfurized):

a PS-GCTCGTTGCGGGACTTAACCCAACAT ("26-mer") The phosphorothioate analogof the multimer moved very closely in gel electrophoresis to thecorresponding normal multimer.

EXAMPLE 3

The procedure of Example 1 was repeated except using as a sulfurizationreagent, 0.2M sulfur in 50% CS₂ /50% triethylamine ("TEA"), tosuccessfully produce PS-TTTT.

EXAMPLE 4

The procedure of Example 2 was repeated except using as a sulfurizationreagent 0.2M sulfur in 50% CS₂ /50% TEA. In both this Example and inExample 3, the respective sulfurized nucleotide multimers were obtained.However, the sulfurization reagent of Examples 1 and 2 is preferred overthat of this Example and Example 3, since it was found that when TEA wasused as the tertiary amine rather than DIEA, a brown glue like substancestarted appearing in the flask after about 6 hours, which was not thecase when DIEA was used (the S/CS₂ /DIEA showing no signs of instabilityeven after 3 days).

EXAMPLE 5

The procedure of Examples 1 and 2 were repeated to again successfullyproduce PS-TTTT, and a (the 26-mer), but using the methoxy, rather thanthe β-cyanoethoxy, phosphoramidites of the nucleosides of the formula:##STR2##

EXAMPLE 6

The procedures of Examples 3 and 4 were repeated to again successfullyproduce the PS-TTTT and a (the 26 mer), but using the methoxyphosphoramidites of Example 5.

EXAMPLE 7

A sulfurization reagent was prepared consisting of 0.2M S in 25% CS₂/25% pyridine/50% TEA. The procedure of Example 1 was then repeatedexcept using the foregoing sulfurization reagent. The procedure ofExample 2 was also repeated, to prepare a (the 26-mer), using theforegoing sulfurization reagent. In addition, using the foregoingsulfurization reagent and the procedure of Example 2, the followingnucleotide multimers were also successfully prepared. ##STR3##

EXAMPLE 8

The procedure of Example 7 was repeated, but using the methoxyphosphoramidites of the nucleotides, to successfully prepare thesulfurized oligomers a, b, c, and d.

EXAMPLE 9

The procedure of Example 1 was again essentially repeated except usingthe methyl, rather than the β-cyanoethoxy, phosphonamidite of theT-nucleoside having the formula: ##STR4## In addition, the sulfurizationreagent used was 0.2M sulfur in 25% CS₂ /25% pyridine/50% TEA. Further,in order to cleave the PS-nucleotide from the resin, the resin wastreated at room temperature with 1 ml of a 1:1 (by volume) mixture ofethylenediamine/EtOH for 6 hours.

The resulting product was PS-TTTT in which the nucleosides are linked bymethyl phosphonodiester groups.

EXAMPLE 10

The procedure of Example 2 was essentially repeated to prepare thesulfurized 15-mer of c, except using the methyl phosphonamidite and thecleavage procedure Example 9. The resulting oligomer has the sequence cin which the nucleosides are linked by methyl phosphonothioate diestergroups.

EXAMPLE 11

Example 9 was repeated except the sulfurizing reagent used as 0.2Msulfur in 50% CS₂ /50% DIEA (which as previously mentioned is preferredover that using TEA, or that in which pyridine is additionally present).The resulting product was again the PS-TTTT oligomer in which thenucleotides are linked by methyl phosphonothioate diester groups of theformula: ##STR5##

EXAMPLE 12

The table below compares the Tm of two phosphorothioate analogs withnormal nucleotide multimers. The P³² labeled nucleotide multimer washybridized with E. coli RNA in 0.48M phosphate buffer, the hybrid boundto hydroxyapatite resin and the thermal stability measured in 0.1Mphosphate buffer.

    ______________________________________                                        Tm °C.                                                                                    Phosphoro-                                                             Normal thioate                                                                Multimer                                                                             Multimer                                                   ______________________________________                                        15-mer        41.8     40.3                                                   26-mer        66.1     50.9                                                   ______________________________________                                    

The foregoing Examples illustrate specific applications of theinvention. Other useful applications of the invention which may be adeparture from the specific Examples will be apparent to those skilledin the art. Accordingly, the present invention is not limited to thoseexamples described above.

I claim:
 1. A composition comprising a solution of sulfur withoutheating, a first nucleoside phosphite or nucleoside phosphonite linkedto a second nucleotide, and a trialkyl amine sized to allow thesulfurization in an organic solvent able to solubilize sulfur.
 2. Acomposition as defined in claim 1 wherein the solvent is carbondisulfide.
 3. A composition as defined in claim 1 or 2 wherein the firstnucleoside phosphite or nucleoside phosphonite is bound to a solid phasesupport.
 4. A composition as defined in claim 3 wherein the solid phasesupport is controlled pore glass.
 5. A composition comprising a solutionof sulfur, a first nucleoside phosphite or nucleoside phosphonite linkedto second nucleotide, diisopropylethylamine, and an organic solvent ableto solubilize sulfur, wherein said first nucleotide phosphite ornucleoside phosphonite is bound to a solid phase support on an automatednucleotide multimer synthesizer and the composition undergoessulfurization of said phosphite or phosphonite in about 45 secondswithout heating.
 6. A composition as defined in claim 5 wherein theorganic solvent is carbon disulfide.
 7. A composition as defined inclaim 5 or 6 wherein the solid phase support is controlled pore glass.